Structural and physiological evidence suggest strongly that the interdigitating microtubules (MTs) of the interpolar mitotic spindle slide over one another during anaphase spindle elongation. Preliminary work implies that spindles contain an enzyme which resembles dynein, the MT-binding ATPase from flagella which promotes sliding between axonemal doublet MTs. We will test the hypothesis that a dynein-like enzyme is important for mitotic motions by studying such molecules isolated from the eggs of sea urchins and nematodes. Antibodies have been raised to polypeptides of sea urchin dynein; these will be used to localize dynein by immunocytochemistry in mitotic cells. Antibodies that interfere with dynein functions in vitro, such as MT binding, ATPase activity, and axoneme beating, will be injected into living cells to test the importance of particular dynein functions for both mitosis and ciliary action in vivo. We have identified a high molecular weight, MT-binding polypeptide in eggs of the nematode Caenorhabditis elegans, an organism that never forms motile cilia. This polypeptide co-electrophoreses with the heavy chain of Tetrahymena dynein and is released from MTs by high salt or Mg-ATP. Preliminary evidence suggests that it copurifies on gel filtration columns with a Mg-ATPase activity. We will test the hypothesis that it is a functionally significant component of the mitotic spindle by the strategy sketched above. We will compare the nematode protein with axonemal dyneins and other MT-associated proteins by enzymatic, immunological and physical chemical criteria. We have also identified a prevalent 134 kD polypeptide in extracts of sea urchin eggs that binds quantitatively to MTs in the presence of the nonhydrolyzable ATP analogue, AMPPNP. Like dynein, it is released quantitatively from the MTs by high salt or Mg-ATP. Rabbit antibodies to electrophoretically-purified material bind in immunoblots to a peptide of the same molecular weight from isolated spindles. It is our working hypothesis that this protein is related to the factor in squid axoplasm that moves particles over the surface of MTs. We suppose that it contributes to the forces that pull chromosomes to the poles by sliding MTs through a spindle matrix. We will test this hypotheses in much the same way we are studying dynein. We will also work with these enzymes to try to reconstruct aspects of mitotic movements in vitro. Through this combination of biochemistry and cell biology, we hope to learn about both the functions and the mechanisms of these putative mitotic motors.